Confirmation of natural hybrids between Gentiana straminea and G. siphonantha (Gentianaceae) based on molecular evidence

A few individuals with intermediate morpho-logy always appeared in the sympatric distributions of Gentiana straminea and G. siphonantha. These intermedi-ate individuals were hypothesized to be the hybrids of two species after a careful evaluation of their morphological characteristics. To test this hypothesis, sequence compar-ison of the internal transcribed spacer (ITS) regions of the nuclear ribosomal and trnS (GCU)-trnG (UCC) inter-genic spacer region of the chloroplast DNA from Gentiana straminea, G. siphonantha and the putative hybrids was performed. The results suggest that most intermediate individuals were the natural hybrids between G. straminea and G. siphonantha. In addition, we exam-ined the sequence variation among the individuals of both parent species and analyzed the possibility leading to the incongruent identification in some individuals based on morphologic and molecular evidences, respectively. The intraspecific diversification of DNA fragments within both parent species and their high variability in hybrid swarms probably resulted from chloroplast genome recombination and incomplete lineage sorting during the early stages of speciation origin of the parent species.     

Phylogenetic origin of Beckmannia （Poaceae） inferred from molecular evidence

The phylogenetic origin of Beckmannia remains unknown. The genus has been placed within the Chlorideae, Aveneae （Agrostideae）, Poeae, or treated as an isolate lineage, Beckmanniinae. In the present study, we used nuclear internal transcribed spacer （ITS） and chloroplast trnL-F sequences to examine the phylogenetic relationship between Beckmannia and those genera that have assumed to be related. On the basis of the results of our studies, the following conclusions could be drawn： （i） Beckmannia and Alopecurus are sister groups with high support; and （ii） Beckmannia and Alopecurus are nested in the Poeae clade with high support. The results of our analysis suggest that Beckmannia should be placed in Poeae.     

Sensitive and rapid detection of Karenia mikimotoi (Dinophyceae) by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) assay was developed to detect the genomic DNA of Karenia mikimotoi using a set of four specific primers based on a ribosomal DNA internal transcribed spacer (ITS). The sensitivity of this LAMP assay was 100-fold higher than regular PCR, and its specificity was validated using other algae as a comparison. Two visual detection approaches were feasible to interpret the positive or negative results. This technology may have the potential to aid in forecasting red-tides on the scene because of its high sensitivity, specificity and rapid detection.     

基于分子证据确认秦岭藤属与驼峰藤属(夹竹桃科)的系统位置

秦岭藤属(Biondia Schltr.)是中国特有属,约13种,驼峰藤属(Merrillanthus Chun&Tsiang)是仅在我国与柬埔寨分布的单种属,目前两个属均被归并到白前属(Vincetoxicum Wolf),由于取样和系统发育分析尚存一些欠缺,因此其系统位置和归属尚需进一步确认。该文使用2个核糖体基因序列片段(ITS、ETS)、5个叶绿体基因序列片段(psbA-trnH、trnG、trnL、trnL-F、trnT-L),以及二者的合并数据,重建娃儿藤亚族(Tylophorinae)(包含属的模式秦岭藤[B.chinensis Schltr.=V.shaanxiense(Schltr.) Meve&Liede]与驼峰藤[M.hainanensis Chun&Tsiang=V.hainanense(Chun&Tsiang) Meve, H. H. Kong&Liede])的系统发育树。结果表明：秦岭藤属与驼峰藤属均包含在白前属中,秦岭藤与青龙藤[B.henryi(Warb.) Tsiang&Li=V.henryi(Warb....     

Reproductive ecology of the Qinghai-Tibet Plateau endemic Gentiana straminea (Gentianaceae), a hermaphrodite perennial characterized by herkogamy and dichogamy

The combined occurrence of both herkogamy and dichogamy in a hermaphrodite species has been considered to strongly favour outcrossing. In this study, we investigated in detail the reproductive ecology of Gentiana straminea Maxim. (Gentianaceae), a hermaphrodite perennial endemic to the Qinghai-Tibet Plateau. In a series of observations and experiments over four consecutive years, we examined whether the combination of dichogamy and herkogamy in individual flowers completely prevents geitonogamous pollen transfer in this species. The mode of floral development clearly indicates that autonomous self-pollination is completely avoided through herkogamy and dichogamy in individual flowers. This implication was confirmed by the breeding experiments, since no seed was produced when flowers were isolated. However, this gentian proved to be highly self-compatible when geitonogamous selfing was artificially induced. Many flowers opened simultaneously on individual plants, the ratio of male to female phase flowers was close to 2:1 in each inflorescence at the full anthesis phase, and they were randomly distributed amongst the upper, middle and lower parts of each stem's inflorescence. On average, Bombus sushikini Skorikov, the most frequent visitor and only legitimate pollinator of G. straminea, visited nearly two flowers per inflorescence, and four flowers per plant. Among the pollinators' foraging bouts, the proportions of geitonogamous visits to inflorescences or flowers within an individual plant were 29% and 37%, respectively. Therefore, despite the strict dichogamous and herkogamous characteristics of the individual flowers, geitonogamous selfing might still prevail in G. straminea because of the size of its floral displays and the continuous visiting behavior of B. sushkini.     

Morphological and molecular evidence of natural hybridization between two distantly related Rhododendron species from the Sino-Himalaya

Rhododendron (Ericaceae) is a large woody genus in which hybridization may play an important role in evolution and speciation, particularly in the Sino-Himalayan region, where many interfertile species often occur sympatrically. Natural hybridization between Rhododendron delavayi Franch. (=  R. arboreum ssp. delavayi ) and Rhododendron decorum Franch., which belong to different subsections of subgenus Hymenanthes, was investigated. Material of R. delavayi and R. decorum and their putative hybrids was collected from the wild. On the basis of morphology, chloroplast DNA, nuclear ribosomal DNA, and AFLP profiles, hybrids and parental species were identified. Hybridization occurred in both directions, but was asymmetrical, with R. delavayi as the major maternal parent in the hybrid zone. Most of the hybrids possessed intermediate phenotypes, and amongst the 15 hybrids detected were six F1s, two F2s, one first-generation backcross to R. delavayi , and two first-generation backcrosses to R. decorum . This indicates that, if Rhododendron underwent rapid radiation in this region, it did so in spite of permeable species barriers.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 156 , 119–129.     

A phylogeny of Packera (Senecioneae; asteraceae) based on internal transcribed spacer region sequence data and a broad sampling of outgroups

A phylogeny of Packera is presented based on sequence data from the internal transcribed spacer region of nuclear ribosomal DNA of 26 species (28 populations) of Packera and 23 outgroup taxa, including representatives of all three subtribes of the Senecioneae. The results support a Mexican origin for Packera, with its closest relatives found among Old World taxa in the subtribe Senecioninae, such as Senecio jacobaea and Pericallis. Packera species from the west coast of the United States, previously included in the section Bolanderi of Greenman, are part of a basal assemblage including species of Greenman's Mexican section Sanguisorboidei. The rest of Packera separates into two sister groups, one containing species from the Arizona-New Mexico region and the other containing more geographically diverse taxa. Among the outgroups, New World Senecio species are monophyletic and two Tussilaginoid assemblages are strongly supported; the Tephroseroid group (Tephroseris and Sinosenecio) plus Petasites combine with the Luina complex to form a clade of north temperate taxa, and the four Mexican genera (Psacalium, Robinsonecio, Barkleyanthus, and Pittocaulon) form a monophyletic group.     

Phylogenetic analysis of the genus Hexachlamys (Myrtaceae) based on plastid and nuclear DNA sequences and their taxonomic implications

Myrtaceae are one of the most species‐rich families of flowering plants in the Neotropics. They include several complex genera and species; Hexachlamys is one of the complex genera. It has not been recognized as a distinct genus and has been included in Eugenia, based on morphological grounds. Therefore, molecular systematic studies may be useful to understand and to help to solve these relationships. Here, we performed a molecular phylogenetic analysis using plastid and nuclear data in order to check the inclusion of Hexachlamys in Eugenia. Plastid (accD, rpoB, rpoC1, trnH‐psbA) and nuclear (ITS2) sequence data were analysed using Bayesian and maximum parsimony methods. The trees constructed using ITS2 and trnH‐psbA were the best able to resolve the relationships between species and genera, revealing the non‐monophyly of Hexachlamys. The molecular phylogenetic analyses were in agreement with previous morphological revisions that have included Hexachlamys in Eugenia. These results reinforce the importance of uniting knowledge and strategies to understand better issues of delimitation of genera and species in groups of plants with taxonomic problems. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172 , 532–543.     

Phylogenetic relationships within Pappophoreae s.l. (Poaceae: Chloridoideae): Additional evidence based on ITS and trnL-F sequence data

Historically, Pappophoreae included the genera Cottea, Enneapogon, Kaokochloa, Pappophorum and Schmidtia. Some authors consider this tribe as a well-supported monophyletic group; while other evidences reveals Pappophoreae as polyphyletic, with Pappophorum separated from the rest of the tribe. When the latter happens, it can form a clade with Tridens flavus. Molecular phylogenetic analyses of the subfamily Chloridoideae have included few species of Pappophoreae; therefore, further research involving more representatives of this tribe is needed. With the aim of providing new evidence to help clarify the phylogenetic position of Pappophorum and its relationships with other genera of the tribe and the subfamily Chloridoideae, eight new sequences of ITS and trnL-F regions of Pappophoreae species were generated. These sequences were analyzed together with other available sequence data obtained from GenBank, using maximum parsimony and Bayesian inference, for individual (trnL-F or ITS) or combined trnL-F/ITS data sets. All analyses reveal that Pappophoreae is polyphyletic, with Pappophorum separated from the rest of the tribe forming a well-supported clade sister to Tridens flavus.     

ITS polymorphism within a single strain ofSclerotium rolfsii

Two morphologically distinct strains, 63–76 and 63H1, were isolated from a protoplast and a hyphal tip of the parentalSclerotium rolfsii strain S-63, respectively. Strains 63–76 and 63H1 showed reduced mycelial growth and lacked clamp connections on hyphae. The two strains also differed from each other and from their parent in RAPD patterns generated by several primers, suggesting that 63–76 and 63H1 were homokaryons isolated from the hetereokaryon S-63. Whereas the parent S-63 belonged to ITS-RFLP group 1, RFLP patterns of internal transcribed spacer (ITS) regions of rDNA of 63–76 and 63H1 were similar to those of ITS-RFLP groups 5 and 3, respectively. The sequence similarity of ITS regions were more than 99% between 63–76 and group 5 strains, 100% between 63H1 and the group 3 strain, and 96.3% between 63–76 and 63H1. Direct sequencing failed in the parental strain S-63. S-63 was considered to contain ITS types of groups 5 and 3.     

Phylogeny of the GenusTrichodermaBased on Sequence Analysis of the Internal Transcribed Spacer Region 1 of the rDNA Cluster

Sequences of the internal transcribed spacer region 1 (ITS1) of the ribosomal DNA were used to determine the phylogenetic relationships of species ofTrichodermasect.Pachybasium.To this end, 85 strains—including all the availableex-type strains—were analyzed. Parsimony analysis demonstrated that the section is nonmonophyletic, distributing the 85 strains among three main groups that were supported by bootstrap values. Group A comprises two clades (A1 and A2), with A1 includingT. polysporum, T. piluliferum,andT. minutisporum,while A2 includedT. hamatum, T. pubescens,andT. strigosumin addition to species previously included in sect.Trichoderma(i.e.,T. viride, T. atroviride,andT. koningii). Theex-type strain ofT. fasciculatumformed a separate branch basal to clade A. Clade B contained the sect.PachybasiummembersT. harzianum, T. fertile, T. croceum, T. longipile, T. strictipile, T. tomentosum, T. oblongisporum, T. flavofuscum, T. spirale,and the anamorphs ofHypocrea semiorbisandH. cf. gelatinosa.Sequence differences among clades A1, A2, and B were in the same order of magnitude as between each of them andT. longibrachiatum,which was used as an outgroup in these analyses. Sequence differences within clades A1, A2, and B were considerably smaller: in some cases (i.e.,T. virensandT. flavofuscum; T. strictipileandH. cf. gelatinosa), the ITS1-sequences were identical, suggesting conspecifity. In other cases (e.g.,T. crassumandT. longipile; T. harzianum, T. inhamatum, T. croceum, T. fertile,andH. semiorbis; T. hamatumandT. pubescens;andT. viride, T. atroviride,andT. koningii) differences were in the range of 1–3 nt only, suggesting a very close phylogenetic relationship. The sequence of a previously described aggressive mushroom competitor group ofT. harzianumstrains (Th2) was strikingly different from that of theex-type strain ofT. harzianumand closely related species and is likely to be a separate species.     

The biogeography of the austral, subalpine genus Ourisia (Plantaginaceae) based on molecular phylogenetic evidence: South American origin and dispersal to New Zealand and Tasmania

Molecular phylogenetic analyses of 26 of the 28 species of Ourisia , including eight of ten subspecies and two purported natural hybrids, are presented and used to examine the biogeography of the genus, which is distributed in subalpine to alpine habitats of South America, New Zealand and Tasmania. Gondwanan vicariance, often cited as the cause of this classic austral biogeographical pattern, was rejected by parametric bootstrapping of our combined dataset. Alternatively, various lines of evidence are presented in favour of a South American origin of Ourisia and subsequent dispersal to Australasia. Specifically, the genus likely arose in the Andes of central Chile and spread to southern Chile and Argentina, to the north-central Andes, and finally to Tasmania and New Zealand. The ancestor of the New Zealand species probably first arrived on the South Island, where the New Zealand species of Ourisia are most diverse, and migrated to the North and Stewart Islands. Because the Tasmanian and New Zealand species are sister to one another, the direction of dispersal between these two areas is equivocal. These results agree with other molecular phylogenetic studies that show that past dispersal between southern hemisphere continents has played an important role in the evolutionary history of many high-elevation austral plants. Our data also show that within South America, many of the geographical barriers (with the exception of the Atacama Desert) that have played a role in the evolution of other plant groups have not affected Ourisia species. Within New Zealand, the phylogeny and biogeography of species of Ourisia coincide with the geological history of the country and patterns of other alpine plants. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 87 , 479–513.     

Phylogeny of Rubus (rosaceae) based on nuclear ribosomal DNA internal transcribed spacer region sequences

We used nuclear ribosomal DNA internal transcribed spacer region (ITS 1 - 5.8S - ITS 2; ITS) sequences to generate the first phylogeny of Rubus based on a large, molecular data set. We sampled 57 taxa including 20 species of subgenus Rubus (blackberries), one to seven species from each of the remaining 11 subgenera, and the monotypic and closely related Dalibarda. In Rubus, ITS sequences are most informative among subgenera, and variability is low between closely related species. Parsimony analysis indicates that Rubus plus Dalibarda form a strongly supported clade, and D. repens may nest within Rubus. Of the subgenera with more than one species sampled, only subgenus Orobatus appears monophyletic. Three large clades are strongly supported: one contains all sampled species of nine of the 12 subgenera; another includes extreme Southern Hemisphere species of subgenera Comaropsis, Dalibarda, and Lampobatus; and a third clade consists of subgenus Rubus plus R. alpinus of subgenus Lampobatus. Rubus ursinus appears to be a hybrid between a close relative of R. macraei (subgenus Idaeobatus, raspberries) and an unidentified subgenus Rubus species. ITS sequences are generally consistent with biogeography and ploidy, but traditionally important morphological characters, such as stem armature and leaf type, appear to have limited phylogenetic value in Rubus.     

Genotyping of Pneumocystis carinii f. sp. hominis isolates in Japan based on nucleotide sequence variations in internal transcribed spacer regions of rRNA genes

Genotyping of Pneumocystis carinii (Pc) isolated from 24 bronchoalveolar lavage (BAL) fluid specimens in Japan was examined based on nucleotide sequence variations in internal transcribed spacer regions 1 and 2 (ITS1 and ITS2, respectively) of rRNA genes. We found 11 ITS1 genotypes including 2 novel ones and 11 ITS2 genotypes including 3 new ones. Combining the ITS1 and ITS2 genotypes resulted in 30 ITS genotypes, of which 10 are newly described in this report. Two or more genotypes in ITS regions in a specimen were observed in 16 of 24 patients. Our results will be of help for the epidemiological investigation of Pc infection.     

麻花秦艽种子休眠机理及其破除方法

通过对干燥贮藏后熟麻花秦艽种子透水性和粗提物活性的测定以及采用高锰酸钾、赤霉素、青霉素、硫酸、流水浸泡方法进行发芽试验,探讨了种子休眠机理和破除休眠的技术。结果表明:麻花秦艽种子吸水过程符合Logistic曲线,其种皮对种子吸水无阻碍作用;硫酸和高锰酸钾处理极显著提高了麻花秦艽种子的发芽率(P<0.01),说明种皮机械障碍是种子萌发的因素之一。种子粗提物在浓度为0.02—0.2 g/mL时具有很强的抑制活性,均对小麦、白菜和麻花秦艽种子萌发和生长产生抑制作用以及流水浸泡可以提高麻花秦艽种子发芽率,表明种子内源抑制物是影响其休眠的另一因素。不同处理均打破了种子休眠,显著提高了种子发芽率,其中1.5%高锰酸钾浸泡10 min光照培养下种子发芽率及发芽势、发芽指数和活力指数均显著高于其他处理(P<0.05)。相比对照,1.5%高锰酸钾处理的种子发芽率极显著提高106.9%。经1.5%高锰酸钾浸泡10 min的麻花秦艽种子粗提物对白菜种子萌发的抑制作用低于未处理,以浓度为0.02—0.04 g/mL时抑制显著,说明高锰酸钾可减弱麻花秦艽种子内源抑制物的活性。综上所述,麻花秦艽种子的休眠属综合休眠。     

Systematic positions of Lamiophiomis and Paraphlomis (Lamiaceae) based on nuclear and chloroplast sequences

Genera Lamiophlomis and Paraphlomis were originally separated from genus Phlomis s.l. on the basis of particular morphological characteristics. However, their relationship was highly contentious, as evidenced by the literature. In the present paper, the systematic positions of Lamiophlomis, Paraphlomis, and their related genera were assessed based on nuclear internal transcribed spacer (ITS) and chloroplast rpl16 and trnL-F sequence data using maximum parsimony (MP) and Bayesian methods. In total, 24 species representing six genera of the ingroup and outgroup were sampled. Analyses of both separate and combined sequence data were conducted to resolve the systematic relationships of these genera. The results reveal that Lamiophlomis is nested within Phlomis sect. Phlomoides and its genetic status is not supported. With the inclusion of Lamiophlomis rotata in sect. Phlomoides, sections Phlomis and Phlomoides of Phlomis were resolved as monophyletic. Paraphlomis was supported as an inde-pendent genus. However, the resolution of its monophyly conflicted between MP and Bayesian analyses, suggesting the need for expended sampling and further evidence.     

Genetic relationship of Tricholoma matsutake and T. nauseosum from the Northern Hemisphere based on analyses of ribosomal DNA spacer regions

The genetic relationship among Tricholoma matsutake and T. nauseosum strains collected from various parts of the Northern Hemisphere was investigated using sequence analysis of the rDNA ITS region and PCR-RFLP analysis of the rDNA IGS-1 region. ITS sequence similarity between T. matsutake and T. nauseosum ranged between 98.1% and 100%. The strains of T. matsutake from coniferous forests and those from broad-leaved forests showed more than 99.8% similarity in their ITS sequences. Three distinct RFLP types were detected when IGS-1 regions were digested with Cfr13I. RFLP patterns showed no variability among the strains of T. nauseosum and those of T. matsutake from broad-leaved forests. This pattern corresponded to the dominant RFLP type in the Japanese population of T. matsutake. Thus, strains belonging to this RFLP type are widely distributed throughout East Asia and Europe and associated with many tree species of Pinaceae and Fagaceae. The result suggests that T. matsutake in coniferous and broad-leaved forests and T. nauseosum should be treated as the same species genetically.     

川东獐牙菜(龙胆科)的胚胎学研究

利用石蜡切片技术,对川东獐牙菜(Swertia davidii)的胚胎发育过程进行显微观察,并根据现有资料,对獐牙菜属的几种植物进行了比较胚胎学研究。结果表明:川东獐牙菜花药四室,药壁发育为基本型,绒毡层异型起源,为腺质绒毡层,发育后期药室内观察到的绒毡层核是早期该层细胞有丝分裂凸入药室并原位退化形成的,中层细胞3层,药室内壁退化,花药壁表皮宿存,细胞柱状伸长,纤维状加厚;小孢母细胞减数分裂为同时型,四分体排列方式主要为四面体形和左右对称型,少数为"T"形和十字交叉形,成熟花粉为2-细胞类型;子房2心皮、1室,侧膜胎座;薄珠心,单珠被,倒弯生胚珠,大孢子母细胞减数分裂形成4个大孢子直线形排列,合点端的大孢子具功能,雄配子体发育为蓼型;2个极核在受精前融合为1次生核,合点端3个反足细胞宿存,每个细胞均多核和异常膨大,形成明显的反足吸器,并在胚乳之外形成染色较深的类似"外胚乳"结构;珠孔受精,受精作用属于有丝分裂前类型;胚乳发育为核型,胚胎发育为茄型;果实成熟时,种子发育至心形胚阶段;反足细胞在龙胆科一些短命植物中的宿存与分裂具有重要的生殖适应与进化意义。     

基于ITS序列探讨杜鹃属的亚属和组间系统关系

首次报道了 15种杜鹃属 (Rhododendron)植物、1种杜香属 (Ledum)植物和Cassiopefastigiata的内转录间隔区(ITS) (包括 5 .8S)序列。加上从GenBank下载的 13种杜鹃属植物和Bajiariaracemosa的ITS序列 ,以C .fastigiata和B .racemosa为外类群 ,用最大简约法对杜鹃属的亚属和组间的系统关系进行了分析。结果表明 :1)杜鹃属是一个单系类群 ,叶状苞亚属为杜鹃属的基部类群 ;2 )杜香属确应归并到杜鹃属中 ,且与有鳞杜鹃亚属有较近的亲缘关系 ;3)有鳞杜鹃亚属和杜香构成一个单系分支 ,该分支是其余无鳞杜鹃花的姐妹群 ;4 )由无鳞杜鹃花组成的一个分支的内部支持率较低 ,其中常绿杜鹃亚属和映山红亚属均为内部支持率很高的单系类群 ,而羊踯躅亚属和马银花亚属均为多系类群 ;5 )在马银花亚属中 ,长蕊杜鹃组和马银花组均分别得到强烈支持 ,马银花组与异蕊杜鹃亚属可能构成姐妹群关系 ,异蕊杜鹃亚属和马银花组组成的一个分支可能与映山红亚属构成姐妹群关系。